EXTRACELLAR RNA COMPONENT SUMMARY Extracellular RNAs (exRNAs) have been found in all tested human biofluids, and there is increasing evidence that they play a role in pain mediation and modulation, and can serve as biomarkers for identification of those at risk for transitioning to chronic pain after an acute pain episode. This project is the exRNA Component of the Omics Data Generation Center (ODGC) for the Acute to Chronic Pain Signatures (A2CPS) Program. In this Program, the Clinical Centers will recruit and collect clinical data and biofluid samples from two longitudinal cohorts of 1800 subjects each. Biofluid samples will be collected 0, 3, and 6 months after an acute pain episode, consisting of a specific surgical procedure or a specific musculoskeletal trauma. These samples will be used to generate multi-omic data to validate 40 primary outcome biomarkers indicating susceptibility or resilience to development of chronic pain, as well as to identify new candidate biomarkers. For the proposed exRNA Component, Aim 1, which will be executed in Year 1, will involve close collaboration with other components of the A2CPS Program to establish the final study design and protocols. All of the A2CPS Program investigators will work together to establish the 40 primary outcome biomarkers. The ODGC and Clinical Center investigators will jointly decide on the specific sample type(s) and collection/processing/storage methods. The ODGC and Data integration Resource Center/Data Coordination Component (DIRC/DCC) investigators will establish Metadata and Data Standards and a workflow for submission of metadata and data to the DCC. The exRNA Component and the Administrative Core of the ODGC will establish a LIMS for sample and data tracking, and recording of metadata. The exRNA Component and DIRC/Data Integration and Analysis Component (DIAC) will establish qRT-PCR and small RNAseq data analysis pipelines. Aims 2 and 3 will span Years 2-4, with Aim 2 focused on isolation and qRT-PCR and small RNAseq profiling of exRNA from the ~11,000 biofluid samples that will be collected by the Clinical Centers. Aim 3 will encompass submission of metadata and data to the DIRC/DCC, quality control of the data, and data analysis and interpretation. The primary goal of this Component is generation and submission of high-quality exRNA qRT-PCR and small RNAseq data for validation of pre- selected candidate exRNA biomarkers and discovery of novel exRNA biomarkers. The exRNA component investigators also anticipate participating in integrative analyses with the DIRC/DIAC aimed at developing pain signatures comprised of multiple biomarker types (including molecular, clinical, psychosocial, and/or imaging biomarkers) indicating susceptibility/resilience to chronic pain, which can be used to develop personalized strategies for prevention and treatment of chronic pain. The personnel for this proposed exRNA Component have acquired the necessary skills and knowledge to successfully execute this project from participation in the Extracellular RNA Communication Consortium, including high-throughput workflows for extracellular vesicle- specific and total exRNA isolation and small RNAseq library preparation and exRNA data analysis pipelines.